A Novel Norditerpene from the Caribbean Sea Plume

J. Org. Chem. 1996, 61, 4487-4490

A Novel Norditerpene from the Caribbean Sea Plume Pseudopterogorgia acerosa (Pallas)

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presence of a R,R'-disubstituted β-carbomethoxyfuran constellation.

Abimael D. Rodrı´guez* and Javier J. Soto Department of Chemistry, University of Puerto Rico, P.O. Box 23346, U.P.R. Station, Rı´o Piedras, Puerto Rico 00931-3346 Received October 27, 1995

Caribbean sea plumes (gorgonians) of the genus Pseudopterogorgia have been shown to produce a variety of chemically interesting and biologically significant secondary metabolites.1 In 1982, Fenical and Clardy examined the constituents of Floridian specimens of Pseudopterogorgia acerosa, resulting in the isolation of pseudopterolide (1), a remarkable metabolite based on the 12-membered carbocyclic pseudopterane skeleton.2 Further examination of extracts of several Pseudopterogorgia spp. has since resulted in the isolation of other pseudopterane metabolites, many of which possess chemically unique structural features.3 Tobagolide (2), one

such metabolite, is a rare nitrogen-containing diterpenoid isolated from a Trinidadian specimen of P. acerosa.4,5 Recently, we have examined the constituents of Puerto Rican specimens of this animal and now report on the isolation and structure determination of alanolide (3), a novel tetracyclic norditerpene which appears to be biogenetically related to tobagolide. The gorgonian octocoral Pseudopterogorgia acerosa was collected by hand using SCUBA (-15 m) at La Parguera, Lajas, Puerto Rico. The CHCl3/CH3OH (1:1) soluble material obtained from the thawed animal was concentrated by rotary evaporation. Solvent partitioning of the crude extract concentrated metabolites into the CHCl3 fraction whose NMR spectra displayed resonances characteristic of pseudopterolide (1).2 Additional purification of the pseudopterane-rich fraction by a combination of silica gel CC and normal-phase HPLC (Partisil 10 M9/ 50, 30% 2-propanol in hexane) afforded 1 as a major constituent (yield ) 0.003%, wet weight) accompanied by 3 (yield ) 0.0002%, wet weight). Compound 3, isolated as a colorless oil (11.6 mg), was clearly unrelated to 1 as its 1H NMR showed a (CH3)2NCH moiety but not the * To whom correspondence should be addressed. Tel: (787) 7640000 ext. 4799. Fax: (787) 751-0625. E-mail: A Rodriguez@ UPR1.UPR.CLU.EDU. (1) Fenical, W. J. Nat. Prod. 1987, 50, 1001-1008. (2) Bandurraga, M. M.; Fenical, W.; Donovan, S. F.; Clardy, J. J. Am. Chem. Soc. 1982, 104, 6463-6465. (3) Rodrı´guez, A. D. Tetrahedron 1995, 51, 4571-4618. (4) Tinto, W. F.; Chan, W. R.; Reynolds, W. F.; McLean, S. Tetrahedron Lett. 1990, 31, 465-468. (5) Chan, W. R.; Tinto, W. F.; Laydoo, R. S.; Machand, P. S.; Reynolds, W. F.; McLean, S. J. Org. Chem. 1991, 56, 1773-1776.

S0022-3263(95)01920-7 CCC: $12.00

A most unusual feature of the 1H NMR (CDCl3) spectrum of alanolide (3) was a nine-proton band at δ 2.35 due to three overlapped methyl groups (one vinyl and two methylamino) and the absence of a three-proton singlet near δ 3.80 characteristic of a β-carbomethoxyfuran moiety. The two additional 1H NMR signals at δ 2.01 (s, 3H) and 1.84 (s, 3H) clearly indicated that like tobagolide 3 contained five methyl groups. The 13C NMR, APT, and 1H-13C COSY (J ) 140 Hz) spectra run in CDCl3 solution revealed a total of 27 protons attached to carbons (6CH, 3CH2, and 5CH3) plus seven quaternary carbons. Intense peaks observable by HREIMS (50 eV), M+ ) 389.1846, or HRFABMS, [M + Na]+) 412.1736, corresponded to a molecular formula of C21H27NO6 (∆ 2.01 ppm of calcd). The nine degrees of unsaturation required by the molecular formula could be ascribed to three carbonyls (13C NMR: δ 204.6, 201.1, 171.4), two double bonds (three CH0 type: 155.8, 143.1, 127.4; and one CH2 type: 113.4), leaving four rings present in the molecule. The IR (neat) spectrum showed no hydroxyl absorptions6 but contained three carbonyl bands at 1785, 1719, and 1639 cm-1. The latter, together with the UV absorption at λmax 260 ( 11 300) nm, indicated the presence of an R,β-unsaturated carbonyl residue. As alanolide (3) could not be obtained in a crystalline state suitable for X-ray crystal structure determination, unequivocal assignment of its structure was performed by 1H and 13C NMR studies (Table 1). Two-dimensional NMR experiments of COSY, long-range COSY, and NOESY were widely used to established scalar and dipolar 1H-1H connectivities. 1H-13C correlations were obtained with HMQC and HMBC experiments (Table 1).7 Detailed analyses of these spectra led to assignments of proton connectivities for three distinct 1H spin systems, subunits 3a-c (Figure 1). The two ketone units 3a,b and the nitrogen-containing γ-lactone unit 3c were connected together in the proper sequence by HMBC data. Confirmation of the structures of the three units as well as the sequencing was provided by HREIMS data (see Scheme 1). The proton-proton connectivities progressing outward from the pivotal H1 resonance (δ 3.27) were easily tracked in the ketone subunit 3a. There were extensive couplings to the neighboring protons H2Rβ, H12 and to (6) Electron impact mass spectrometry of an aliquot of alanolide that had been treated with BSTFA at 25 °C (to make a trimethylsilyl derivative), with introduction by direct inlet probe (DIP), showed that 3 remains underivatized as no active functional groups are present on this molecule. (7) (a) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 20932094. (b) Bax, A.; Aszalos, A.; Dinya, Z.; Sudo, K. J. Am. Chem. Soc. 1986, 108, 8056-8063.

© 1996 American Chemical Society

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Table 1.

1H

NMR (300 MHz),

position

δH, mult, intrgt (J, Hz)

1 2R β 3 4 5R β 6 7 8 9 10 11 12 13 14R β Me15 16 Me17 Me18 19 N(CH3)2

3.27, m, 1H 2.87, t, 1H (6.9) 2.87, t, 1H (6.9) 4.46, dd, 1H (5.7, 8.7) 2.99, dd, 1H (13.2, 8.1) 2.73, dd, 1H (13.2, 6.5) 5.12, dd, 1H (0.9, 6.6) 3.35, d, 1H (6.6) 3.44, d, 1H (0.9) 4.76, dd, 1H (1.5, 4.5) 4.97, br d, 1H (0.6) 4.85, br d, 1H (1.2) 1.84, s, 3H 2.35, s, 3He 2.01, d, 3H (0.6) 2.35, s, 6Hf

13C

Notes

NMR (75 MHz), 1H-1H COSY, NOESY, and HMBC Spectral Data of Alanolide (3) in CDCl3a δC (mult)b

42.8 (d) 45.2 (t) 204.6 (s) 76.9 (d) 44.3 (t) 201.1 (s) 127.4 (s) 78.5 (d) 70.7 (d) 59.8 (s) 60.8 (d) 81.6 (d) 143.1 (s) 113.4 (t) 23.1 (q) 155.8 (s) 20.6 (q) 24.8 (q) 171.4 (s) 40.9 (q, 2X)

1H-1H COSY

NOESY

H2, H12, H14β H1 H1

H2β, H12, Me-15 H11, H14β, Me-15 H1, H14β, Me-15

H5Rβ H4, H5β H4, H5R

H5β H5R

H9, Me-17, Me-18 H8

Me-18 H11d

H12 H1, H11

H2R, H9d, H14β, Me-15, N(CH3)2 H1, H14β, Me-15, N(CH3)2

H14β, Me-15 H1, H14R, Me-15 H14Rβ

H14β, Me-15 H2Rβ, H11, H12, H14R H1, H2Rβ, H11, H12, H14R

H8, Me-18 H8, Me-17

Me-18 H8, Me-17

HMBCc H2Rβ,11,12,14Rβ,15 H1, 4, 12 H1, 2Rβ, 4, 5β H2Rβ, 5Rβ H4 H4, 5Rβ, 8 H5Rβ, 8, 9, 17, 18 H9 H8, 11 H8, 9, 11, 12 H1, 9, 12 H1, 2Rβ, 4, 11 H1, 2Rβ, 12, 14Rβ, 15 H1, 15 H1, 14Rβ H8, 17, 18 H18 H17 H8, 9, 11

H11, H12

a

Chemical shift values are in ppm relative to TMS. Spectra were recorded at room temperature. b 13C NMR multiplicities were obtained by attached proton test (APT) sequences. c Protons correlated to carbon resonances in 13C column. Parameters were optimized for JCH ) 6 and 8 Hz. d This strong NOE was observed in Bz-d6 solution only. e This overlapped signal appears at δ 2.00 (d, 3H, J ) 1.5 Hz) in Bz-d6 solution. f This overlapped signal appears at δ 2.28 (s, 6H) in Bz-d6 solution.

Scheme 1. Analysis of Key HREIMS Fragments

Figure 1. Partial structures of alanolide (3) deduced from HETCOR and 1H-1H COSY.

the more remote exomethylene H14β proton. In turn, H12 showed coupling to the C11 methine as revealed by a small cross peak in the 1H-1H COSY spectrum. The H1-H2Rβ and the H11-H12 coupling responses, however, led to termination points that could not be linked to other spin systems through the normal COSY experiment alone. The observed lack of a spin coupling pathway between either H2R or H2β and H4 was consistent with the separation of these two spin systems by the C3 quaternary carbon. Experimental evidence supporting the C1-C5 connectivity was provided by HMBC data. These results included correlations of the protons H2Rβ with carbon resonances at δ 204.6 (s, C3), 143.1 (s, C13), 76.9 (d, C4), and 42.8 (d, C1). The H5Rβ protons at δ 2.99 and 2.73, respectively, which were shown by COSY to be coupled only to H4, were in turn correlated through HMBC experiments to carbons C3, C4, C6, and C7. Moreover, a long-range HMBC correlation was observed between H4 at δ 4.46 and C12 at δ 81.6, implicating the presence of an ether bridge between C4 and C12. Thus, the COSY and HMBC spectra showed connectivity between the oxygen-bearing carbons, C4 and C12, via the ether bridge and the OdCCH2CH- substructure, which completed the connectivity around the

tetrahydropyran ring. Detection of the COSY and longrange heteronuclear NMR correlation data was consistent with the linking of the C4-C6 and C1-C2 partial substructures through C3. These HMBC correlations were used to join subunit 3b with the upper end of fragment 3a. The connectivity at the lower end of

Notes

fragment 3a in the proton COSY spectrum ended at the oxygen-substituted methine proton (H11, δ 3.44). An HMBC connectivity was observed between H11 and a quaternary oxygen-bearing carbon at a chemical shift of δ 59.8. The carbon chemical shifts of δ 59.8 (s, C10) and 60.8 (d, C11) were found to be shifted upfield with respect to the normal oxygen-bearing carbons, which suggested an epoxide structure. These results, coupled with the HMBC and 1H-1H COSY results, conclusively established the position of the epoxide moiety in alanolide at these carbon positions. The second ketone unit in alanolide was deduced to be a trisubstituted R,β-unsaturated ketone (3b), and the third subunit was shown to possess a nitrogen-containing γ-lactone group (3c). The UV absorption at λmax 260 nm and several redundant HMBC and 1H NMR correlations (Table 1) were used for the mapping of subunit 3b. An observation regarding fragment 3c that was at first perplexing concerned the CH group with NMR chemical shifts of δ 70.7 (d)/3.35 (d) in the range consistent with an oxygen attachment. This group was eventually concluded to be attached to nitrogen and part of a dimethylamino residue based on long-range HMBC correlations from it (H9) to C7/C8/C10/C11/C19 as shown in Table 1. Direct 1H-1H connectivity linking H8 with the aminomethine proton H9 (resonating at 3.35 ppm) was clearly observed from the COSY spectrum. The site of lactone attachment was speculated to be C8, the methine proton of which was apparently shifted downfield to δ 5.12 by virtue of it also being allylic. The presence of a distinct HMBC cross peak observed between the lactone carbonyl carbon (δ 171.4) and H8 confirmed this assignment. The HMBC spectrum also showed the connection between the C19 carbonyl and C9 methine proton. Fragment 3b and fragment 3c were connected in the following manner. Direct protoncarbon connectivity information linking C6 through C9 with branching C16 array was obtained from HMBC data. The H8 oxymethine proton at δ 5.12 showed correlations to the carbon signals ascribed to C6 (201.1, s), C7 (127.4, s), C9 (70.7, d), and C16 (155.8, s). Evidence also supporting the linking of the isopropylidene group to C8 through C7 was provided by long-range protonproton coupling between the Me-17 and Me-18 groups with the H8 proton. Moreover, the lack of coupling between H5R or H5β and H8 was consistent with their being separated by quaternary carbons. Substructure 3c and the lower end of fragment 3a were assembled on the basis of the following evidence. Since the H8/H9 coupling response showed no further correlations to link to the other spin systems in the molecule and clear HMBC correlations were observed from H11 of 3a to C9 (70.7, d) and C19 (171.4, s) of 3c, the C9 through C11 with branching C19 substructure accounted for the remaining atoms. The signals at δ 171.4 (s), 78.5 (d), 70.7 (d), 59.8 (s), and 40.9 (q, 2X) in the 13C NMR spectrum, coupled with the IR absorption at 1785 cm-1, and 1H NMR peaks at δ 5.12 (dd, 1H, J ) 0.9, 6.6 Hz), 3.35 (d, 1H, J ) 6.6 Hz), and 2.35 (s, 6H) were assigned to an R,R,β-trisubstituted γ-lactone functionality resembling that found in 2.4,5 However, the 13C NMR lines observed at δ 59.8 (s) and 60.8 (d) and the broad doublet in the 1H NMR spectrum at δ 3.44 (1H, J < 1Hz) indicated that unlike 2 there is a spiro R-epoxy γ-lactone moiety present in 3. Combinations of the fragments a, b, and c to give 3 is the only one compatible with the characteristics of the remaining atoms (C5, C6, C7, C8, C10, and C11), all of

J. Org. Chem., Vol. 61, No. 13, 1996 4489 Chart 1. Relative Stereochemistry for Alanolide (3) Proposed from NOESY Data

which are devoid of mutual proton-proton couplings. Moreover, since a Dreiding model of alanolide shows that an oxygen bridge between C5 and C12 cannot be connected the only possible formula is 3. Additional support for this overall structure was provided by the HREIMS spectral fragmentation pattern summarized in Scheme 1. The gross structure of alanolide (3) was further supported by the NOESY data. The relative stereochemistry of 3 containing seven chiral centers was deduced from combination of the NOESY data with the 1H-1H coupling constants as shown in Table 1. The 3-oxotetrahydropyran ring was in a chair conformation due to the steric hindrance imposed by the cis-axial orientation between C5 and C11 and the strain of a 10-membered ring (Chart 1). The angular hydrogens H4 and H12 of the tetrahydropyran ring were suggested to be cis equatorial since a characteristic NOESY correlation was observed for H12/H1 with no cross-peaks between H4/H12 or H11/ H12. The spatial relationships of the γ-lactone, epoxide, and tetrahydropyran rings were further confirmed from NOESY cross-peaks for the H9/H11 pair and H2R/H11 in 3. The strong NOE response between the H9 proton and the epoxymethine proton H11 (observed only in C6D6 solution) confirmed the R orientation of the epoxide with H11 cis to C9. Close examination of a Dreiding model of 3 indicated that the C10-C11 epoxide could not adopt a β configuration; to do so would introduce a significant amount of ring strain. The proposed relative stereochemistry was also strongly supported by the 1H-1H coupling (4.5 Hz) between H1 (axial) and H12 (equatorial), suggesting that these two protons were cis. Moreover, the near absence of coupling (J ) 0.9 Hz) of the δ 3.44 absorption for H11 was attributed to the combined electronegativity effects of vicinal trans-coplanar oxygen atoms on the coupling strength of the H11 and H12 protons (Chart 1).8 The dihedral angles between these protons diminish the coupling strength of each proton, reducing their mutual coupling to less than 1 Hz. Alanolide (3) is the first norpseudopterane natural product and possesses a hitherto unknown carbon framework as it lacks the methyl (or carbomethoxyl) carbon normally found at C4 in the regular pseudopterane skeleton. However, the structure of alanolide is similar in many respects to those of tobagolide (2) and the isogersemolides9,10 except that the Me-16 carbon is miss(8) Booth, H. Tetrahedron Lett. 1965, 7, 411-416.

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Notes

ing and that 3 contains an unusual C4,12 oxabridge together with a unique spiro R-epoxy γ-lactone moiety. Tobagolide (2) appears to be biogenetically related to 3. A plausible pathway to explain the biogenetic cleavage of the C4 to C16 bond in 2 is depicted in Scheme 2 (supporting information). It is possible that pseudopteranes are excreted by P. acerosa for chemoprotection; however, the potentially reactive functionality present does not confer cytotoxicity for 1 or 3. Neither pseudopterolide nor alanolide was active against three human tumor cell lines as, they exhibited no detectable cytotoxicity at 50 µg/mL in any of the cell lines (HCT 116, CCRF-CEM and MCF-7). On the other hand, compound 1 proved inactive in the NCI test for agents active against the human immunodeficiency virus (HIV). More detailed studies of the bioactivities of these compounds, along with structural data for related components of the P. acerosa extract, will be reported in due course.

in hexane) afforded 200 mg of known pseudopterolide (1).2 Fraction IX (2.3 g) was separated into nine subfractions over silica gel with 20% acetone in hexane. Subfraction 6 (183 mg) was purified further by HPLC [Partisil 10 M9/10 silica gel with 30% 2-propanol in hexane] to yield 11.6 mg of pure alanolide (3). Alanolide (3): colorless oil; IR (neat) 3081, 3018, 2963, 2942, 2789, 1785, 1719, 1639, 1439, 1372, 1279, 1235, 1211, 1110, 1083, 1052, 993, 754 cm-1; UV (CHCl3) λmax 260 ( 11 300) nm; [R]25D -84.5° (c 2.0, CHCl3); 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3, 75 MHz) (see Table 1); HREI-MS m/z [M+] calcd for C21H27NO6 389.1838, found 389.1846 (17), 333.1576 (6, C18H23NO5), 318.1670 (1, C18H24NO4), 278.1400 (2, C15H20NO4), 208.1349 (11, C12H18NO2), 193.1102 (5, C11H15NO2), 180.1389 (1, C11H18NO), 178.0871 (19, C11H16NO), 165.1157 (21, C10H15NO), 155.0596 (4, C7H9NO3), 150.0923 (13, C9H12NO), 137.0607 (10, C8H9O2), 126.0556 (100, C6H8NO2), 124.1128 (51, C8H14N), 98.0609 (29, C5H8NO), 86.0610 (16, C4H8NO), 85.0539 (34, C4H7NO), 82.0661 (15, C5H8N), 71.0735 (15, C4H9N), 57.0578 (25, C3H7N); HRFAB-MS m/z [M + Na]+ calcd for C21H27NO6Na 412.1735, found 412.1720.

Experimental Section

Acknowledgment. The assistance of Dr. Paul Yoshioka, Dr. Anna Boulanger, Mr. Anthony Sostre, and Miss Ana E. Pomales in specimen collection is gratefully acknowledged. We extend our sincere appreciation to Miss Ileana Rodrı´guez for assistance during the isolation procedures and to Dr. Louis R. Barrows (University of Utah Center for Drug Discovery) for cytotoxicity assays on human cells. The authors thank Dr. John P. Bader (NCI) for the in-vitro anti-HIV activity tests. HREIMS and HRFABMS spectral determinations were performed by the Midwest Center for Mass Spectrometry at the University of Nebraska-Lincoln, a National Science Foundation Regional Facility (Grant No. CHE8211164). The authors acknowledge Dr. Jose´ A. Prieto and Mr. Jose´ R. Martı´nez from the Industry/ University Materials Characterization Center for the determination of 500-MHz NMR spectra. The purchase of the Bruker DRX-500 spectrometer at U.P.R. was made possible thanks to a grant from FOMENTO. This study was supported by the NSF-EPSCoR (Grant No. R118610677), NIH-MBRS (Grant No. S06RR08102-17), and NSF-MRCE (Grant No. R11-8802961) Programs of the University of Puerto Rico at Rı´o Piedras.

General Experimental Procedures. The infrared spectrum (IR) was recorded on a Nicolet 600 FT-IR spectrophotometer. 1H and 13C NMR spectra were measured on a General Electric Multinuclear QE-300; 1H NMR chemical shifts are recorded with respect to the residual CHCl3 signal (7.26 ppm), and 13C NMR chemical shifts are reported in ppm relative to CDCl3 (77.0 ppm). HMQC and HMBC data were recorded on a Bruker DRX-500 spectrometer. The optical rotation was determined on a Perkin-Elmer polarimeter Model 243B. Column chromatography was performed on silica gel (35-75 mesh), and TLC analyses were carried out using glass-packed precoated silica gel plates. High-performance liquid chromatography (HPLC) was done using columns of 10 µm silica gel. All solvents used were either spectral grade or were distilled from glass prior to use. Collection and Extraction of P. acerosa. The Caribbean sea plume P. acerosa (Pallas) was collected by hand using SCUBA at depths of 5-10 m in December 1994 from La Parguera, Lajas, Puerto Rico. A voucher specimen is stored at the Chemistry Department of the University of Puerto Rico. The wet animal (6.7 kg) was blended with MeOH-CHCl3 (1:1) (6 × 1 L), and after filtration, the crude extract was evaporated under vacuum to yield a green residue (634 g). After the crude oil was partioned between hexane and H2O, the aqueous suspension was extracted with CHCl3. The resulting extract was filtered and concentrated in vacuo to yield 46.8 g of a dark orange residue. The pseudopterane-rich extract was chromatographed over silica gel (2.5 kg) and separated into fractions I-XIII on the basis of TLC analyses. Subsequent purification of fraction VII (1.2 g) by column chromatography over silica gel (55.0 g, 15% acetone (9) Williams, D.; Andersen, R. J.; Van Duyne, G. D.; Clardy, J. J. Org. Chem. 1987, 52, 332-335. (10) Williams, D. E.; Andersen, R. J.; Kingston, J. F.; Fallis, A. G. Can. J. Chem. 1988, 66, 2928-2934.

Supporting Information Available: Copies of the HREIMS, 1H NMR (300 MHz), and 13C NMR (75 MHz) spectra for compound 3 plus Scheme 2 (8 pages). This material is contained in libraries on microfiche, immediately follows this article in the microfilm version of this journal, and can be ordered from the ACS; see any current masthead page for ordering information. JO951920G